Bioactive molecules that are isolated from plants, bacteria, and fungi are often referred to as natural products. These molecules are synthesized by primary or secondary pathways within the organism or may even be degradation products of another molecule. Many of these molecules have shown a variety of therapeutic uses in humans and other animal species. One of the best known examples is taxol, which was originally isolated from the bark of the Pacific Yew tree. Taxol has been shown to have anti-cancer properties and is currently used in the treatment of breast cancer. Actinomycetes are prolific producers of bioactive small molecules. These molecules may be used chemically as immunosuppressants, antibiotics, and cancer therapeutics. Actinomycetes are Gram-positive bacteria that form long, thread-like branched filaments. The term actinomycetes is used to indicate organisms belonging to Actinomycetales, an Order of the domain Bacteria. The Actinomycetales are divided into 34 Families including Streptomyceteae, to which belongs the Genus Streptomyces (Bergey's Manual of Systematic Bacteriology, Second Edition, 2001; George M. Garrity, Editor-in-Chief, Springer Verlag, New York).
Natural products derived from microbial sources primarily belong to three metabolic families: peptides, polyketides, and terpenes. Peptide natural products can be further classified based on their mode of synthesis: ribosomal and non-ribosomal. Non-ribosomal peptides are synthesized on enzymatic thiotemplates termed non-ribosomal peptide synthetases (NRPS). The non-ribosomal peptides encompass a wide range of compounds having diverse activities including, but not limited to, immunosupressive (such as cyclosporin), surfactant (such as surfactin), siderophores (such as enterobactin), virulence factors (such as yersinabactin), antibacterial (such as penicillin and vancomycin), and anti-cancer (such as actinomycin and bleomycin) activities (Weber et al., Current Genomics 1994; 26:120–25; Ehmann et al., Proc. Nat. Acad. Sci. 2000; 97:2509–14; Gehring et al., Biochemistry 1998; 37:11637; Kallow et al., Biochemistry 1998; 37:5947–52; Trauger et al., Proc. Nat. Acad. Sci. 2000; 97:3112–17; Schauweker et al., J. Bacteriology 1999; 27:2468–74; and Shen et al., Bioorganic Chem 1999; 27:155–71). Non-ribosomal peptides typically range in size from 1–11 amino acids and are produced by a variety of microbes including cyanobacteria, actinomycetes and fungi.
In many cases the non-ribosomal peptides contain non-proteogenic amino acids such as norleucine, β-alanine, ornithine, etc., for which biogenesis pathways, which are secondary to primary metabolism, are required and are post-synthetically modified (e.g., hydroxylated or methylated) by tailoring enzymes. As used herein, the term “proteogenic” indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways. The choice of including a (D)- or (L)-amino acid into a peptide of the present invention depends, in part, on the desired characteristics of the peptide. For example, the incorporation of one or more (D)-amino acids can confer increasing stability on the peptide in vitro or in vivo. As used herein, the term “amino acid equivalent” refers to compounds which depart from the structure of the naturally occurring amino acids, but which have substantially the structure of an amino acid, such that they can be substituted within a peptide that retains biological activity. Thus, for example, amino acid equivalents can include amino acids having side chain modifications and/or substitutions, and also include related organic acids, amides or the like. The term “amino acid” is intended to include amino acid equivalents. The term “residues” refers both to amino acids and amino acid equivalents.
The genes required to make a NRPS and the necessary tailoring enzymes have been shown in all cases to be localized to the chromosome of the producing microbe. NRPSs are modular in nature, where a module may be defined as a segment of the NRPS necessary to catalyze the activation of a specific amino acid and result in the incorporation of that amino acid into a non-ribosomal peptide. A minimal module contains three domains: (1) adenylation domains (about 60 kDa), responsible for selecting and activating an amino acid and transferring the aminoacyl adenylate to a peptidyl carrying center; (2) thiolation domains, also referred to as peptidyl carrier proteins (8–10 kDa), containing a serine residue which is post-translationally modified with a 4-phosphopantetheine group (Ppant) which acts as an acceptor for the aminoacyl adenylate; and (3) condensation domains (50–60 kDa) which catalyze peptide bond-forming chain-translocating steps between an upstream peptidyl-s-Ppant and the downstream aminoacyl-Ppant of the adjacent module (Doekel, S. and Marahiel, M. A. 2000; Chem. Biol. 7:373–384). This minimal module for chain extension is typically repeated within a synthetase and a co-linear relationship exists between the number of modules present and the number of amino acids in the final product with the order of the modules in the synthetase determining the order of the amino acids in the peptide.
There is a continuing need in the art to determine the genes encoding NRPS complexes.